Utilizing primary HLA-G+ extravillous trophoblasts and HLA-G+ EVT-like cell lines to study maternal-fetal interactions.
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SUBMITTER: Hamilton I
PROVIDER: S-EPMC10200998 | biostudies-literature | 2023 May
REPOSITORIES: biostudies-literature
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Utilizing primary HLA-G+ extravillous trophoblasts and HLA-G+ EVT-like cell lines to study maternal-fetal interactions.
Hamilton Ira I Ikumi Nadia M NM Kshirsagar Sarika S Goodman Wendy A WA Tilburgs Tamara T
STAR protocols 20230511 2
Fetal extravillous trophoblasts (EVTs) are the most invasive cells of the placenta and play a key role in modulating maternal immune responses. Here, we present a protocol to purify and culture human leukocyte antigen-G (HLA-G)+ EVTs. We describe steps for tissue dissection, tissue digestion, density gradient centrifugation, and cell sorting, and we provide detailed methods to determine EVT function. HLA-G+ EVTs are isolated from two maternal-fetal interfaces: the chorionic membrane and the basa ...[more]
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Project description:Seven patient paired primary human HLA-G+ extravillous trophoblasts (EVT) and Villous trophoblasts (VT) obtained from 1st trimester (7-9 weeks) villous tissue were obtained. RNA was isolated directly after isolation and purification using FACS sort for CD45-HLA-G+ (EVT) and CD45-HLA-G-EGFR1+ (VT) fractions. Expression profiles were compared to two samples of the choriocarcinoma cell line JEG-3 and four samples of decidual stromal cells (DSC) at passage 2 after cell culture.
2015-05-15 | E-MTAB-3217 | biostudies-arrayexpress
Project description:IntroductionCirculating fetal extravillous trophoblasts may offer a superior alternative to cell-free fetal DNA for noninvasive prenatal testing. Cells of fetal origin are a pure source of fetal genome; hence, unlike the cell-free noninvasive prenatal test, the fetal cell-based noninvasive prenatal test is not expected to be affected by maternal DNA. However, circulating fetal cells from previous pregnancies may lead to confounding results.Material and methodsTo study whether fetal trophoblast cells persist in maternal circulation postpartum, blood samples were collected from 11 women who had given birth to a boy, with blood sampling at 1-3 days (W0), 4-5 weeks (W4-5), around 8 weeks (W8) and around 12 weeks (W12) postpartum. The existence of fetal extravillous trophoblasts was verified either by X and Y chromosome fluorescence in situ hybridization analysis or by short tandem repeat analysis. To exclude technological bias in isolating fetal cells, blood samples were also collected from 10 pregnant women between a gestational age of 10 and 14 weeks, the optimal time frame for cell-based noninvasive prenatal test sampling. All the samples were processed according to protocols established by ARCEDI Biotech for fetal extravillous trophoblast enrichment and isolation.ResultsFetal extravillous trophoblasts were found in all the 10 samples from pregnant women between a gestational age of 10 and 14 weeks. However, only 4 of 11 blood samples taken from women at 1-3 days postpartum rendered fetal extravillous trophoblasts, and only 2 of 11 samples rendered fetal extravillous trophoblasts at 4 weeks postpartum.ConclusionsIn this preliminary dataset on few pregnancies, none of the samples rendered any fetal cells at or after 8 weeks postpartum, showing that cell-based noninvasive prenatal testing based on fetal extravillous trophoblasts is unlikely to be influenced by circulating cells from previous pregnancies.
| S-EPMC7384123 | biostudies-literature
Project description:During pregnancy, invading HLA-G+ extravillous trophoblasts (EVT) play a key role in placental development, uterine spiral artery remodeling, and prevention of detrimental maternal immune responses to placental and fetal antigens. Failures of these processes are suggested to play a role in the development of pregnancy complications, but very little is known about the underlying mechanisms. Here we present validated methods to purify and culture primary HLA-G+ EVT from the placental disk and chorionic membrane from healthy term pregnancy. Characterization of HLA-G+ EVT from term pregnancy compared to first trimester revealed their unique phenotypes, gene expression profiles, and differing capacities to increase regulatory T cells (Treg) during coculture assays, features that cannot be captured by using surrogate cell lines or animal models. Furthermore, clinical variables including gestational age and fetal sex significantly influenced EVT biology and function. These methods and approaches form a solid basis for further investigation of the role of HLA-G+ EVT in the development of detrimental placental inflammatory responses associated with pregnancy complications, including spontaneous preterm delivery and preeclampsia.
| S-EPMC7355041 | biostudies-literature
Project description:Invading human leukocyte antigen-G+ (HLA-G+) extravillous trophoblasts (EVT) are rare cells that are believed to play a key role in the prevention of a maternal immune attack on foreign fetal tissues. Here highly purified HLA-G+ EVT and HLA-G- villous trophoblasts (VT) were isolated. Culture on fibronectin that EVT encounter on invading the uterus increased HLA-G, EGF-Receptor-2, and LIF-Receptor expression on EVT, presumably representing a further differentiation state. Microarray and functional gene set enrichment analysis revealed a striking immune-activating potential for EVT that was absent in VT. Cocultures of HLA-G+ EVT with sample matched decidual natural killer cells (dNK), macrophages, and CD4+ and CD8+ T cells were established. Interaction of EVT with CD4+ T cells resulted in increased numbers of CD4+CD25(HI)FOXP3+CD45RA+ resting regulatory T cells (Treg) and increased the expression level of the Treg-specific transcription factor FOXP3 in these cells. However, EVT did not enhance cytokine secretion in dNK, whereas stimulation of dNK with mitogens or classical natural killer targets confirmed the distinct cytokine secretion profiles of dNK and peripheral blood NK cells (pNK). EVT are specialized cells involved in maternal-fetal tolerance, the properties of which are not imitated by HLA-G-expressing surrogate cell lines.
| S-EPMC4466754 | biostudies-literature
Project description:OBJECTIVES:During human pregnancy, the endothelial cells of the uterine spiral arteries (SPA) are extensively replaced by a subtype of placental trophoblasts, endovascular extravillous trophoblasts (enEVTs), thus establishing a placental-maternal circulation. On this pathway, foetus-derived placental villi and enEVTs bath into the maternal blood that perfuses along SPA being not attacked by the maternal lymphocytes. We aimed to reveal the underlying mechanism of such immune tolerance. METHODS:In situ hybridization, immunofluorescence, ELISA and FCM assay were performed to examine TGF-β1 expression and distribution of regulatory T cells (Tregs) along the placental-maternal circulation route. The primary enEVTs, interstitial extravillous trophoblasts (iEVTs) and decidual endothelial cells (dECs) were purified by FACS, and their conditioned media were collected to treat naïve CD4+ T cells. Treg differentiation was measured by FLOW and CFSE assays. RESULTS:We found that enEVTs but not iEVTs or dECs actively produced TGF-β1. The primary enEVTs significantly promoted naïve CD4+ T-cell differentiation into immunosuppressive FOXP3+ Tregs, and this effect was dependent on TGF-β1. In recurrent spontaneous abortion (RSA) patients, an evidently reduced proportion of TGF-β1-producing enEVTs and their ability to educate Tregs differentiation were observed. CONCLUSIONS:Our findings demonstrate a unique immune-regulatory characteristic of placental enEVTs to develop immune tolerance along the placental-maternal circulation. New insights into the pathogenesis of RSA are also suggested.
| S-EPMC7260064 | biostudies-literature
Project description:STUDY QUESTION:Does A Disintegrin And Metalloproteinase 8 (ADAM8) control extravillous trophoblast (EVT) differentiation and migration in early human placental development? SUMMARY ANSWER:ADAM8 mRNA preferentially localizes to invasive HLA-G-positive trophoblasts, associates with the acquirement of an EVT phenotype and promotes trophoblast migration through a mechanism requiring β1-integrin. WHAT IS KNOWN ALREADY:Placental establishment in the first trimester of pregnancy requires the differentiation of progenitor trophoblasts into invasive EVTs that produce a diverse repertoire of proteases that facilitate matrix remodeling and activation of signaling pathways important in controlling cell migration. While multiple ADAM proteases, including ADAM8, are highly expressed by invasive trophoblasts, the role of ADAM8 in controlling EVT-related processes is unknown. STUDY DESIGN, SIZE, DURATION:First trimester placental villi and decidua (6-12 weeks' gestation), primary trophoblasts and trophoblastic cell lines (JEG3, JAR, Bewo, HTR8/SVNeo) were used to examine ADAM8 expression, localization and function. All experiments were performed on at least three independent occasions (n = 3). PARTICIPANTS/MATERIALS, SETTING, METHODS:Placental villi and primary trophoblasts derived from IRB approved first trimester placental (n = 24) and decidual (n = 4) were used to examine ADAM8 localization and expression by in situ RNAScope hybridization, flow cytometry, quantitative PCR and immunoblot analyses. Primary trophoblasts were differentiated into EVT-like cells by plating on fibronectin and were assessed by immunofluorescence microscopy and immunoblot analysis of keratin-7, vimentin, epidermal growth factor receptor (EGFR), HLA-G and ADAM8. ADAM8 function was examined in primary EVTs and trophoblastic cell lines utilizing siRNA-directed silencing and over-expression strategies. Trophoblast migration was assessed using Transwell chambers, cell-matrix binding was tested using fibronectin-adhesion assays, and ADAM8-β1-integrin interactions were determined by immunofluorescence microscopy, co-immunoprecipitation experiments and function-promoting/inhibiting antibodies. MAIN RESULTS AND THE ROLE OF CHANCE:Within first trimester placental tissues, ADAM8 preferentially localized to HLA-G+ trophoblasts residing within anchoring columns and decidua. Functional experiments in primary trophoblasts and trophoblastic cell lines show that ADAM8 promotes trophoblast migration through a mechanism independent of intrinsic protease activity. We show that ADAM8 localizes to peri-nuclear and cell-membrane actin-rich structures during cell-matrix attachment and promotes trophoblast binding to fibronectin matrix. Moreover, ADAM8 potentiates β1-integrin activation and promotes cell migration through a mechanism dependent on β1-integrin function. LIMITATIONS, REASONS FOR CAUTION:The primary limitation of this study was the use of in vitro experiments in examining ADAM8 function, as well as the implementation of immortalized trophoblastic cell lines. Histological localization of ADAM8 within placental and decidual tissue sections was limited to mRNA level analysis. Further, patient information corresponding to tissues obtained by elective terminations was not available. WIDER IMPLICATIONS OF THE FINDINGS:The novel non-proteolytic pro-migratory role for ADAM8 in controlling trophoblast migration revealed by this study sheds insight into the importance of ADAM8 in EVT biology and placental development. STUDY FUNDING/COMPETING INTEREST(S):This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC-Discovery Grant) and the Canadian Institutes of Health Research (CIHR-Open Operating Grant). There are no conflicts or competing interests. TRIAL REGISTRATION NUMBER:NA.
| S-EPMC6154767 | biostudies-literature
Project description:The distinct human leukocyte antigen (HLA) class I expression pattern of human extravillous trophoblasts (EVT) endows them with unique tolerogenic properties that enable successful pregnancy. Nevertheless, how this process is elaborately regulated remains elusive. Previously, E74 like ETS transcription factor 3 (ELF3) was identified to govern high-level HLA-C expression in EVT. In the present study, ELF3 is found to bind to the enhancer region of two adjacent NOD-like receptor (NLR) genes, NLR family pyrin domain-containing 2 and 7 (NLRP2, NLRP7). Notably, our analysis of ELF3-deficient JEG-3 cells, a human choriocarcinoma cell line widely used to study EVT biology, suggests that ELF3 transactivates NLRP7 while suppressing the expression of NLRP2. Moreover, we find that NLRP2 and NLRP7 have opposing effects on HLA-C expression, thus implicating them in immune evasion at the maternal-fetal interface. We confirmed that NLRP2 suppresses HLA-C levels and described a unique role for NLRP7 in promoting HLA-C expression in JEG-3. These results suggest that these two NLR genes, which arose via gene duplication in primates, are fine-tuned by ELF3 yet have acquired divergent functions to enable proper expression levels of HLA-C in EVT, presumably through modulating the degradation kinetics of IkBα. Targeting the ELF3-NLRP2/NLRP7-HLA-C axis may hold therapeutic potential for managing pregnancy-related disorders, such as recurrent hydatidiform moles and fetal growth restriction, and thus improve placental development and pregnancy outcomes.
| S-EPMC11295039 | biostudies-literature
Project description:The number of molecules identified to be involved in communication between placenta and decidua is fast expanding. Previously, we showed that NODAL expressed in maternal endometrial stromal cells is able to affect NODAL and STOX1 expression in placental extravillous trophoblasts. The effect of maternal NODAL on placental NODAL expression is achieved via Activin A, while preliminary data suggests that maternal NODAL affects STOX1 expression in trophoblasts potentially via IGF1. In the current study, T-HESC endometrial stromal cells were treated with siRNAs against NODAL after which IGF1 mRNA expression was determined by quantitative RT-PCR, while IGF1 secretion was measured by ELISA. Recombinant IGF1 and inhibitors of the MAPK and PI3K/AKT pathways were added to SGHPL-5 extravillous trophoblasts after which the effects on STOX1 mRNA and STOX1 protein expression were determined. The effect of IGF1 and the MAPK and PI3K/AKT inhibitors on the invasive capacity of SGHPL-5 cells was investigated by performing invasion assays. We found that T-HESC cells treated with NODAL siRNAs showed significant upregulation of IGF1 mRNA expression and IGF1 protein secretion. Addition of IGF1 to SGHPL-5 cell media significantly upregulated STOX1 mRNA and protein expression. Using inhibitors of the PI3K/AKT and MAPK pathway showed that the effect of IGF1 on STOX1 expression is accomplished via MAPK signaling. Secondly, PI3K inhibition independently leads to reduced STOX1 expression which can be rescued by adding IGF1. IGF1 was unable to influence the invasive capacity of SGHPL-5 cells, while inhibiting the PI3K/AKT pathway did reduce the invasion of these cells. To conclude, here we show that downregulated NODAL expression in endometrial stromal cells, previously associated with pre-eclampsia like symptoms in mice, increases IGF1 secretion. Increased levels of IGF1 lead to increased expression levels of STOX1 in extravillous trophoblasts via the MAPK pathway, hereby identifying a novel signaling cascade involved in maternal-fetal communication.
| S-EPMC6084977 | biostudies-literature
Project description:BackgroundNoninvasive prenatal testing (NIPT) based on cell-free DNA in maternal circulation has been accepted worldwide by the clinical community since 2011 but limitations, such as maternal malignancy and fetoplacental mosaicism, preclude its full replacement of invasive prenatal diagnosis. We present a novel silicon-based nanostructured microfluidics platform named as "Cell Reveal™" to demonstrate the feasibility of capturing circulating fetal nucleated red blood cells (fnRBC) and extravillous cytotrophoblasts (EVT) for cell-based noninvasive prenatal diagnosis (cbNIPD).MethodsThe "Cell Reveal™" system is a silicon-based, nanostructured microfluidics using immunoaffinity to capture the trophoblasts and the nucleated RBC (nRBC) with specific antibodies. The automated computer analysis software was used to identify the targeted cells through additional immunostaining of the corresponding antigens. The identified cells were retrieved for whole genome amplification for subsequent investigations by micromanipulation in one microchip, and left in situ for subsequent fluorescence in situ hybridization (FISH) in another microchip. When validation, bloods from pregnant women (n = 24) at gestational age 11-13+6 weeks were enrolled. When verification, bloods from pregnant women (n = 5) receiving chorionic villus sampling or amniocentesis at gestation age 11+4-21 weeks with an aneuploid or euploid fetus were enrolled, followed by genetic analyses using FISH, short tandem repeat (STR) analyses, array comparative genomic hybridization, and next generation sequencing, in which the laboratory is blind to the fetal genetic complement.ResultsThe numbers of captured targeted cells were 1-44 nRBC/2 ml and 1-32 EVT/2 ml in the validation group. The genetic investigations performed in the verification group confirmed the captured cells to be fetal origin. In every 8 ml of the maternal blood being blindly tested, both fnRBC and EVT were always captured. The numbers of captured fetal cells were 14-22 fnRBC/4 ml and 1-44 EVT/4 ml of maternal blood.ConclusionsThis report is one of the first few to verify the capture of fnRBC in addition to EVT. The scalability of our automated system made us one step closer toward the goal of in vitro diagnostics.
| S-EPMC5712079 | biostudies-literature
Project description:ObjectiveInvasion of uterine spiral arteries by extravillous trophoblasts in the first trimester of pregnancy results in loss of endothelial and musculoelastic layers. This remodeling is crucial for an adequate blood supply to the fetus with a failure to remodel implicated in the etiology of the hypertensive disorder preeclampsia. The mechanism by which trophoblasts induce this key process is unknown. This study gives the first insights into the potential mechanisms involved.Methods and resultsSpiral arteries were dissected from nonplacental bed biopsies obtained at Caesarean section, and a novel model was used to mimic in vivo events. Arteries were cultured with trophoblasts in the lumen, and apoptotic changes in the endothelial layer were detected after 20 hours, leading to loss of endothelium by 96 hours. In vitro, coculture experiments showed that trophoblasts stimulated apoptosis of primary decidual endothelial cells and an endothelial cell line. This was blocked by caspase inhibition and NOK2, a FasL blocking antibody. NOK2 also abrogated trophoblast-induced endothelial apoptosis in the vessel model.ConclusionsExtravillous trophoblast induction of endothelial apoptosis is a possible mechanism by which the endothelium is removed, and vascular remodeling may occur in uterine spiral arteries. Fas/FasL interactions have an important role in trophoblast-induced endothelial apoptosis.
| S-EPMC4228192 | biostudies-literature